Streptococcus pneumoniae causes more fatal infections world-wide than almost any other pathogen (Anonymous, 1991; Fraser, 1982). In the U.S.A., deaths caused by S. pneumoniae rival in numbers those caused by AIDS (Anonymous, 1991). In the U.S.A., most fatal pneumococcal infections occur in individuals over 65 years of age, in whom S. pneumoniae is the most community-acquired pneumonia. In the developed world, most pneumococcal deaths occur in the elderly, or in immunodeficient patents including those with sickle cell disease. In the less-developed areas of the world, pneumococcal infection is one of the largest causes of death among children less than 5 years of age (Berman et al., 1985; Greenwood et al., 1987; Spika et al., 1989, Bale, 1990). The increase in the frequency of multiple antibiotic resistance among pneumococci and the prohibitive cost of drug treatment in poor countries make the present prospects for control of pneumococcal disease problematical (Munoz et al., 1992; Marton et al., Klugman, 1990).
Humans acquire pneumococci through aerosols or by direct contact. Pneumococci first colonize the upper airways and can remain for weeks or months. As many as 50 or more of young children and the elderly are colonized. In most cases, this colonization results in no apparent infection (Gray et al., 1980; Gray et al., 1981; Hendley et al., 1975). Studies of outbreak strains have suggested that even highly virulent strains can colonize without causing disease (Smillie et al., 1938; Smillie et al., 1936; Gratten et al., 1980; DeMaria et al., 1984). These expectations have been recently confirmed using molecular probes to fingerprint individual clones (M. J. Crain, personal communication to one of the inventors). In some individuals, however, the carried organism can give rise to symptomatic sinusitis or middle ear infections. If pneumococci are aspirated into the lung, especially with food particles or mucus, they can cause pneumonia. Infections at these sites generally shed some pneumococci into the blood where they can lead to sepsis, especially if they continue to be shed in large numbers from the original focus of infection. Pneumococci in the blood can reach the brain where they can cause meningitis. Although pneumococcal meningitis is less common than other infections caused by these bacteria, it is particularly devastating; some 10% of patients die and greater than 50% of the remainder have life-long neurological sequelae (Bohr et al., 1985; Klein et al., 1981).
In elderly adults, the present 23-valent capsular polysaccharide vaccine is about 60% effective against invasive pneumococcal disease with strains of the capsular types included in the vaccine (Bolan et al., 1986; Shapiro et al., 1991). The 23-valent vaccine is not effective in children less than 2 years of age because of their inability to elicit adequate responses to most polysaccharides (Cowan et al., 1978; Gotschlich et al., 1977). Improved vaccines that can protect children and adults against invasive infections with pneumococci would help reduce some of the most deleterious aspects of this disease. A vaccine that protected against disease but did not reduce pneumococcal carriage rates would not, however, be expected to control the disease in immunocompromised individuals (Shapiro et al., 1991) and in unimmunized individuals. Such a vaccine would also not be expected to affect the rates of infection in immunized children prior to the development of an adequate antibody or immunological response.
A strategy that could control infections in all of these individuals would be any form of immunization that prevented or greatly reduced carriage, and hence transmission of pneumococci. In the case of immunization of young children with Haemophilus influenzae group b polysaccharide-protein conjugates, it has been observed that carriage is reduced from about 4% to less than 1%, (Barbour et al., 1993), a possible explanation of concomitant herd immunity (Chiu et al., 1994). If a vaccine could prevent colonization by pneumococci, such a vaccine would be expected to prevent virtually all pneumococcal infections in the immunized patients. Since even unimmunized patients must acquire pneumococci from others, a vaccine that reduced carriage should reduce infections in immunocompromised, as well as unimmunized patients. In fact, an aggressive immunization program, coupled with antibiotic treatment of demonstrated carriers, might be able to largely eliminate the human reservoir of this organism. It may not be possible, however, to totally eliminate pneumococci since there are a number of reports that they have been found in laboratory rodents (Fallon et al., 1988). Whether these pneumococci are infectious for man, easily transmittable to man, or even pathogens in wild rodents is not known. S. pneumoniae does not live free in the environment.
Although intramuscular immunization with capsular polysaccharide vaccines has been effective at reducing the incidence of pneumococcal sepsis in the elderly (Shapiro et al., 1991), it has not been reported to affect pneumococcal carriage rates in children up to 54 months of age (Douglas et al., 1986; Douglas et al., 1984). The principal determinant of specific immunity at mucosal surfaces is secretory IgA (S-IgA) which is physiologically and functionally separate from the components of the circulatory immune system. Mucosal S-IgA response are predominantly generated by the common mucosal immune system (CMIS) (Mestecky, 1987), in which immunogens are taken up by specialized lympho-epithelial structures collectively referred to as musoca-associated lymphoid tissue (MALT). The term common mucosal immune system refers to the fact that immunization at any mucosal site can elicit an immune response at all other mucosal sites (Mestecky, 1987). Thus, immunization in the gut can elicit mucosal immunity in the upper airways and visa versa. The best studied MALT structures are the intestinal Peyer's patches (Mestecky, 1987). Further, it is important to note that oral immunization can induce an antigen-specific IgG response in the systemic compartment in addition to mucosal IgA antibodies (McGhee, J. R. and H. Kiyono 1993, "New perspectives in vaccine development: mucosal immunity to infections", Infectious Agents and Disease 2:55-73).
Most soluble and non-replicating antigens are poor mucosal immunogens, especially by the peroral route, probably because they are degraded by digestive enzymes and have little or no tropism for the gut associated lymphoid tissue (GALT). A notable exception is cholera toxin (CT). CT is a potent mucosal immunogen (Elson, 1989; Lycke et al., 1986; Wilson et al., 1989) probably because of the GM1 ganglioside-binding property of its binding subunit, CTB, that enables it to be taken up by the M cells of Peyer's patches and passed to the underlying immunocompetent cells. In addition to being a good mucosal immunogen, CT is a powerful adjuvant which greatly enhances the mucosal immunogenicity of other soluble antigens co-administered with it (Elson, 1989; Lycke et al., 1986; Wilson et al., 1989). Although it remains somewhat controversial, pure or recombinant CTB probably does not have these properties when administered intragastrically (i.g.) as an adjuvant. Very small amounts (&lt;1 .mu.g) of intact CT, however, can act synergistically with CTB as a powerful oral adjuvant (Wilson et al., 1990). This finding may account for apparent adjuvant activity of many commercial preparations of CTB that usually contain small amounts of contaminating CT.
The mechanisms by which CT and CTB act as adjuvants are not fully understood, but are certainly complex, and appear to depend on several factors including 1) the toxic activity associated with the ADT-ribosylating property of the Al subunit (Abbas et al., 1991); 2) increased permeability of mucosae (Abbas et al., 1991; Ziegler-Heitbrock et al., 1992); 3) enhanced antigen-presenting cell function (with increased levels of IL-1) (Abbas et al., 1991; Ziegler-Heitbrock et al., 1992); as well as 4) direct stimulation of T and B cell activities (Elson, 1989; Lycke et al., 1986; Wilson et al., 1989; Wilson et al., 1990). This last point is controversial, however, as the in vitro effects of CT or CTB on T and B cells are generally inhibitory rather than stimulatory (Abbas et al., 1991). Nevertheless, numerous reports attest to the in vivo mucosal immunoenchancing effects of CT and of CTB coupled to antigens (Hakansson et al., 1994; Anderson et al., 1981; Liang et al., 1988; Dagen et al., 1995; Dillard et al., 1994; Szu et al. 1989). Recent studies have shown that orally-administered CT can induce Th2 type responses for antigen-specific serum IgG and mucosal IgA antibodies (Xu-Amano, J., H. Kiyono, R. J. Jackson, H. F. Staats, Fujihashi, P. D. Burrows, C. O. Elson, S. Pillai and J. R. McGhee. 1993, "Helper T cell subsets for immunoglobulin A responses: Oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues", J. Exp. Med. 178:1309-1320, Xu-Amano, R. J. Jackson, K. Fujihashi, H. Kiyono, H. F. Staats and J. R. McGhee, 1994, "Helper Th1 and Th2 cell responses following mucosal or systemic immunization with cholera toxin", Vaccine 12:903-911). Recently Elson et al. have shown that CT selectively inhibits CD8.sup.+ cells, and therefore tends to abrogate suppressive-effects (Elson et al., 1995).
Since immunity to carriage would be expected to operate at the mucosal surface, any attempt to identify antigens for vaccines against carriage should include immunizations designed to elicit mucosal immune responses. Accordingly, the oral (including peroral, intragastric) immunization or administration with pneumococcal proteins, as in the present invention has not, it is believed, been heretofore disclosed or suggested or, in addition, the evaluation of adjuvants, as in the present disclosure, has not, it is believed, been heretofore taught or suggested.